Impact of dietary digestible aromatic amino acid levels and stachyose on growth, nutrient utilization, and cecal odorous compounds in broiler chickens.

This study evaluated the impact of dietary digestible aromatic amino acid (DAAA) levels and stachyose on growth, nutrient utilization and cecal odorous compounds in broiler chickens. A 3×2 two-factor factorial design: Three dietary DAAA levels (1.40, 1.54, 1.68%) supplemented with either 5 g/kg of stachyose or without any stachyose were used to create 6 experimental diets. Each diet was fed to 6 replicates of 10 birds from d 22 to 42. Findings revealed that broilers receiving a diet with 1.54% DAAA levels supplemented with 5 g/kg stachyose exhibited a significant boost in average daily gain and improved utilization of crude protein, ether extract, tryptophan, and methionine compared to other diet treatments (P < 0.05). As the dietary DAAA levels increased, there was a significant rise in the concentrations of indole, skatole, p-methylphenol, and butyric acid in the cecum of broilers (P < 0.05). The addition of stachyose to diets reduced concentrations of indole, skatole, phenol, p-methylphenol, acetic acid and propionic acid in the cecum (P < 0.05). The lowest concentrations of indole, phenol, p-methylphenol, volatile fatty acids and pH in cecum of broilers were observed in the treatment which diet DAAA level was 1.40% with stachyose (P < 0.05). In conclusion, dietary DAAA levels and stachyose had significant interactions on the growth, main nutrient utilization and cecal odorous compounds in broilers. The dietary DAAA level was 1.54% with 5 g/kg of stachyose can improve the growth performance, nutrient utilization. However, the dietary DAAA level was 1.40% with stachyose was more beneficial to decrease the cecal odor compound composition in broilers.

Histoplasma capsulatum Relies on Tryptophan Biosynthesis To Proliferate within the Macrophage Phagosome.

Histoplasma capsulatum yeasts reside and proliferate within the macrophage phagosome during infection. This nutrient-depleted phagosomal environment imposes challenges to Histoplasma yeasts for nutrition acquisition. Histoplasma yeasts require all 20 amino acids, which can be formed by de novo biosynthesis and/or acquired directly from the phagosomal environment. We investigated how Histoplasma obtains aromatic amino acids (i.e., phenylalanine, tyrosine, and tryptophan) within the phagosome during infection of macrophages. Depletion of key enzymes of the phenylalanine or tyrosine biosynthetic pathway neither impaired Histoplasma's ability to proliferate within macrophages nor resulted in attenuated virulence in vivo. However, loss of tryptophan biosynthesis resulted in reduced growth within macrophages and severely attenuated virulence in vivo. Together, these results indicate that phenylalanine and tyrosine, but not tryptophan, are available to Histoplasma within the macrophage phagosome. The herbicide glyphosate, which targets 5-enolpyruvylshikimate-3-phosphate synthase of the aromatic amino acid biosynthetic pathway, inhibited Histoplasma yeast growth, and this growth inhibition was partially reversed by aromatic amino acid supplementation or overexpression of ARO1. These results suggest that the aromatic amino acid biosynthetic pathway is a candidate drug target to develop novel antifungal therapeutics.

Time-restricted feeding affects transcriptomic profiling of hypothalamus in pigs through regulating aromatic amino acids metabolism.

BACKGROUND: Time-restricted feeding (TRF) is an effective means that can efficiently regulate the metabolism and health of animals and humans. However, the effect of TRF on hypothalamic function remains unclear. RESULTS: Results showed that TRF significantly increased the activities of digestive enzymes lipase, maltase in the duodenum and lipase, trypsin in the pancreas whereas significantly decreased serum gastrointestinal hormones gastrin, glucagon-like peptide-1, cholecystokinin, peptide YY, and ghrelin. Metabolites related to amino acid metabolism, including citrulline, kynurenine, N-acetylleucine, l-tryptophan, and l-tyrosine, significantly increased in the TRF group. Differential metabolites were mainly enriched in phenylalanine, tyrosine, and tryptophan biosynthesis and tryptophan metabolism. Transcriptomic analysis of hypothalamus showed that a total of 462 differentially expressed genes (DEGs) were significantly changed by TRF. In particular, DEGs such as DDC, TH, GOT2, and DBH involved in aromatic amino acid metabolism pathways were significantly downregulated, whereas the expression of CYP1B1 was significantly upregulated. Moreover, DEGs (PDYN and PPP3CA) involved in amphetamine addiction and cocaine addiction were also downregulated in the TRF group. CONCLUSION: Taken together, these results suggested that TRF improved the digestion and absorption of nutrients and thus increased the accessibilities of aromatic amino acids. The increasing of circulating aromatic amino acids might mediate the regulatory neuroendocrine effects of TRF regimes on the hypothalamus functions, especially on drug addictions. This study reveals a possible mechanism underlying the effects of regulating feeding patterns on the function of the hypothalamus by altering aromatic amino acids metabolism. © 2022 Society of Chemical Industry.

Berberine alleviates type 2 diabetic symptoms by altering gut microbiota and reducing aromatic amino acids.

OBJECTIVE: Berberine (BBR), which is extracted from traditional Chinese herb, is abundant in Coptis chinensis and Berberis vulgaris, with a treatment on type 2 diabetes mellitus (T2DM). However, its oral bioavailability is poor. Therefore, the ability of BBR to regulate gut microbiota and intestinal metabolites might exist. This study aimed to investigate changes in gut microbiota and intestinal metabolites, and to reveal the potential mechanism of BBR. METHODS: To observe the role of gut microbiota in the treatment of T2DM by BBR, antibiotics intervened gut microbiota was used in this study, and the therapeutic effects of BBR were evaluated. A 16S rRNA gene sequencing approach was utilized to analyze gut microbiota alterations, and UHPLC-QTOF/MS-based untargeted metabolomics analysis of colon contents was used to identity differential intestinal metabolites. Finally, serum aromatic amino acids (AAAs) were absolutely quantified using LC/MS. RESULTS: Inhibition of the blood glucose levels, and improvements in glucose tolerance and serum lipid parameters were observed in the BBR treated group. Type 2 diabetic symptoms in rats in the BA group (treated with antibotics and BBR) were alleviated. However, the therapeutical effects are weaker in the BA group compared with the BBR group, indicating that BBR can be used to treat type 2 diabetic rats immediately, and modulation of gut microbiota is related to the mechanism of BBR in the treatment of T2DM. The community richness and diversity of the gut microbiota were significantly increased by BBR, and the relative abundance of Bacteroidetes was increased in the BBR group, which was accompanied by a decreased relative abundance of Proteobacteria and Verrucomicrobia at the phylum level. At the family level, a probiotic Lactobacillaceae was significantly upregulated not only in the BBR group but also in the BA group and was negatively associated with the risk of T2DM. Metabolomic analysis of colon contents identified 55 differential intestinal metabolites between the BBR group and the model group. AAAs, including tyrosine, tryptophan and phenylalanine, were obviously decreased in the BBR group not only in the colon contents but also in the serum. CONCLUSIONS: These results demonstrated that BBR could alleviate symptoms in type 2 diabetic rats by affecting gut microbiota composition and reducing the concentration of AAAs.

CD98hc (SLC3A2) sustains amino acid and nucleotide availability for cell cycle progression.

CD98 heavy chain (CD98hc) forms heteromeric amino acid (AA) transporters by interacting with different light chains. Cancer cells overexpress CD98hc-transporters in order to meet their increased nutritional and antioxidant demands, since they provide branched-chain AA (BCAA) and aromatic AA (AAA) availability while protecting cells from oxidative stress. Here we show that BCAA and AAA shortage phenocopies the inhibition of mTORC1 signalling, protein synthesis and cell proliferation caused by CD98hc ablation. Furthermore, our data indicate that CD98hc sustains glucose uptake and glycolysis, and, as a consequence, the pentose phosphate pathway (PPP). Thus, loss of CD98hc triggers a dramatic reduction in the nucleotide pool, which leads to replicative stress in these cells, as evidenced by the enhanced DNA Damage Response (DDR), S-phase delay and diminished rate of mitosis, all recovered by nucleoside supplementation. In addition, proper BCAA and AAA availability sustains the expression of the enzyme ribonucleotide reductase. In this regard, BCAA and AAA shortage results in decreased content of deoxynucleotides that triggers replicative stress, also recovered by nucleoside supplementation. On the basis of our findings, we conclude that CD98hc plays a central role in AA and glucose cellular nutrition, redox homeostasis and nucleotide availability, all key for cell proliferation.

Increasing carbohydrate availability in the hindgut promotes hypothalamic neurotransmitter synthesis: aromatic amino acids linking the microbiota-brain axis.

The gut microbiota is increasingly recognized to modulate brain function by recent studies demonstrating the central effects of various gut microbial manipulation strategies. Our previous study demonstrated that antibiotic-induced alterations of hindgut microbiota are associated with changes in aromatic amino acid (AAA) metabolism and hypothalamic neurochemistry, while the underlying mechanistic insight is limited. Given that the microbial AAA metabolism can be affected by luminal carbohydrate availability, here we hypothesize that increasing hindgut carbohydrate availability affects the expression of neurotransmitters in the porcine hypothalamus. A hindgut microbiota-targeted strategy was adopted by increasing hindgut carbohydrate availability in a cecal-cannulated piglet model. Mechanistic involvement of AAAs along the gut microbiota-brain axis was further investigated in mice and neuronal cells. Increasing carbohydrate availability by cecal starch infusion led to a decrease in hindgut AAA metabolism, and an increase in systemic AAA availability, central AAA-derived neurotransmitters (5-HT, dopamine), and neurotrophin BDNF in piglets, indicating that hindgut microbiota affect hypothalamic neurochemistry in an AAA-dependent manner. Single AAA i.p. injection in mice revealed that an increase in circulating tryptophan and tyrosine elevated their concentrations in brain and finally promoted the expressions of 5-HT, dopamine, and BDNF in a time-dependent manner. Neuronal cells treated with single AAAs in vitro further demonstrated that tryptophan and tyrosine enhanced 5-HT and dopamine synthesis, respectively, and promoted BDNF expression partly through the 5-HT1A/DRD1-CREB pathway. Our study reveals that increasing hindgut carbohydrate availability promotes hypothalamic neurotransmitter synthesis and that AAAs act as potential mediators between hindgut microbiota and brain neurochemistry.

Antibiotics-induced modulation of large intestinal microbiota altered aromatic amino acid profile and expression of neurotransmitters in the hypothalamus of piglets.

The evidence of gut microbiota-mediated modulation of brain function has been widely recognized from studies using germ-free rodents or animals with oral antibiotic-induced microbiota depletion. Since the large intestine harbors greater numbers and more diverse of microbes than in the small intestine, large intestinal microbiota may play a crucial role in the modulation of brain function. In this study, a large intestinal microbiota-targeted strategy was used to investigate the impact of large intestinal microbiota on brain function. Twelve piglets (12.08 ± 0.28 kg) fitted with a T-cannula at the distal ileum were fed a standard diet and randomly assigned to two groups (n = 6) for ileal infusion of either saline or antibiotics. After 25 days of infusion, ileal and fecal microbiota, serum amino acids and neurotransmitters, and hypothalamic transcriptomics were analyzed. While the antibiotic infusion did not change the proximal ileal microbial composition, it markedly altered the fecal microbial composition and increased aromatic amino acid (AAAs) metabolism (p < 0.05), suggesting the infusion specifically targeted large intestinal microbes. Concentrations of AAAs were likewise decreased in the blood and hypothalamus (p < 0.05) by antibiotic infusion. Antibiotic infusion further decreased concentrations of hypothalamic 5-hydroxytryptamine (5-HT) and dopamine, in line with AAAs being their precursors. An up-regulation in gene expressions of neurotransmitter transporters and synthetases was observed (q < 0.001). In conclusion, the distalileal-antibiotic infusion altered neurotransmitter expression in the porcine hypothalamus and this effect occurred simultaneously with changes in both the large intestinal microbiota, and AAAs in the large intestine, blood and hypothalamus. These findings indirectly indicate that large intestinal microbiota affects hypothalamic neurotransmitter expressions. Read the Editorial Highlight for this article on page 208.

Shaofu Zhuyu decoction ameliorates obesity-mediated hepatic steatosis and systemic inflammation by regulating metabolic pathways.

Shaofu Zhuyu decoction (SFZYD, also known as Sobokchugeo-tang), a classical prescription drug in traditional East Asian medicine, has been used to treat blood stasis syndrome (BSS). Hepatic steatosis is the result of excess caloric intake, and its pathogenesis involves internal retention of phlegm and dampness, blood stasis, and liver Qi stagnation. To evaluate the effects of treatment with SFZYD on obesity-induced inflammation and hepatic steatosis, we fed male C57BL/6N mice a high fat diet (HFD) for 8 weeks and then treated them with SFZYD by oral gavage for an additional 4 weeks. The results of histological and biochemical examinations indicated that SFZYD treatment ameliorates systemic inflammation and hepatic steatosis. A partial least squares-discriminant analysis (PLS-DA) scores plot of serum metabolites showed that HFD mice began to produce metabolites similar to those of normal chow (NC) mice after SFZYD administration. We noted significant alterations in the levels of twenty-seven metabolites, alterations indicating that SFZYD regulates the TCA cycle, the pentose phosphate pathway and aromatic amino acid metabolism. Increases in the levels of TCA cycle intermediate metabolites, such as 2-oxoglutaric acid, isocitric acid, and malic acid, in the serum of obese mice were significantly reversed after SFZYD treatment. In addition to inducing changes in the above metabolites, treatment with SFZYD also recovered the expression of genes related to hepatic mitochondrial dysfunction, including Ucp2, Cpt1α, and Ppargc1α, as well as the expression of genes involved in lipid metabolism and inflammation, without affecting glucose uptake or insulin signaling. Taken together, these findings suggest that treatment with SFZYD ameliorated obesity-induced systemic inflammation and hepatic steatosis by regulating inflammatory cytokine and adipokine levels in the circulation and various tissues. Moreover, treatment with SFZYD also reversed alterations in the levels of metabolites of the TCA cycle, the pentose phosphate pathway and aromatic amino acid metabolism.

Overexpression of plastid transketolase in tobacco results in a thiamine auxotrophic phenotype.

To investigate the effect of increased plastid transketolase on photosynthetic capacity and growth, tobacco (Nicotiana tabacum) plants with increased levels of transketolase protein were produced. This was achieved using a cassette composed of a full-length Arabidopsis thaliana transketolase cDNA under the control of the cauliflower mosaic virus 35S promoter. The results revealed a major and unexpected effect of plastid transketolase overexpression as the transgenic tobacco plants exhibited a slow-growth phenotype and chlorotic phenotype. These phenotypes were complemented by germinating the seeds of transketolase-overexpressing lines in media containing either thiamine pyrophosphate or thiamine. Thiamine levels in the seeds and cotyledons were lower in transketolase-overexpressing lines than in wild-type plants. When transketolase-overexpressing plants were supplemented with thiamine or thiamine pyrophosphate throughout the life cycle, they grew normally and the seed produced from these plants generated plants that did not have a growth or chlorotic phenotype. Our results reveal the crucial importance of the level of transketolase activity to provide the precursor for synthesis of intermediates and to enable plants to produce thiamine and thiamine pyrophosphate for growth and development. The mechanism determining transketolase protein levels remains to be elucidated, but the data presented provide evidence that this may contribute to the complex regulatory mechanisms maintaining thiamine homeostasis in plants.

The activity of GAT107, an allosteric activator and positive modulator of α7 nicotinic acetylcholine receptors (nAChR), is regulated by aromatic amino acids that span the subunit interface.

GAT107, the (+)-enantiomer of racemic 4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide, is a strong positive allosteric modulator (PAM) of α7 nicotinic acetylcholine receptor (nAChR) activation by orthosteric agonists with intrinsic allosteric agonist activities. The direct activation produced by GAT107 in electrophysiological studies is observed only as long as GAT107 is freely diffusible in solution, although the potentiating activity primed by GAT107 can persist for over 30 min after drug washout. Direct activation is sensitive to α7 nAChR antagonist methyllycaconitine, although the primed potentiation is not. The data are consistent with GAT107 activity arising from two different sites. We show that the coupling between PAMs and the binding of orthosteric ligands requires tryptophan 55 (Trp-55), which is located at the subunit interface on the complementary surface of the orthosteric binding site. Mutations of Trp-55 increase the direct activation produced by GAT107 and reduce or prevent the synergy between allosteric and orthosteric binding sites, so that these mutants can also be directly activated by other PAMs such as PNU-120596 and TQS, which do not activate wild-type α7 in the absence of orthosteric agonists. We identify Tyr-93 as an essential element for orthosteric activation, because Y93C mutants are insensitive to orthosteric agonists but respond to GAT107. Our data show that both orthosteric and allosteric activation of α7 nAChR require cooperative activity at the interface between the subunits in the extracellular domain. These cooperative effects rely on key aromatic residues, and although mutations of Trp-55 reduce the restraints placed on the requirement for orthosteric agonists, Tyr-93 can conduct both orthosteric activation and desensitization among the subunits.

Metazoan innovation: from aromatic amino acids to extracellular signaling.

Tyrosine depletion in metazoan proteins was recently explained to be due to the appearance of tyrosine kinases in Metazoa. Here, we present a complementary explanation for the depletion of tyrosine, stating the importance of tyrosine in signaling not only as a phosphorylation target but also as a precursor for catecholamines and hormones. Molecules (dopamine, norepinephrine, and epinephrine, and to a lesser extent serotonin and melatonin) critical to metazoan multicellular signaling are also greatly dependent on a supply of tyrosine. These signaling molecules are synthesized in two highly linked pathways specific to metazoans. In addition, the shikimate pathway that non-metazoans use to synthesize the aromatic amino acids is not present in metazoans. These important pathway changes have occurred between Metazoa and other eukaryotes, causing significant changes to tyrosine metabolism and rendering tyrosine crucial for extracellular signaling. In addition, the evolutionary and functional linkage between these two pathways and the resulting implications for neuropathology are discussed.

Metabolic profiling for studying chemotype variations in Withania somnifera (L.) Dunal fruits using GC-MS and NMR spectroscopy.

Withania somnifera (L.) Dunal (Solanaceae), commonly known as Ashwagandha, is one of the most valued Indian medicinal plant with several pharmaceutical and nutraceutical applications. Metabolic profiling was performed by GC-MS and NMR spectroscopy on the fruits obtained from four chemotypes of W. somnifera. A combination of (1)H NMR spectroscopy and GC-MS identified 82 chemically diverse metabolites consisting of organic acids, fatty acids, aliphatic and aromatic amino acids, polyols, sugars, sterols, tocopherols, phenolic acids and withanamides in the fruits of W. somnifera. The range of metabolites identified by GC-MS and NMR of W. somnifera fruits showed various known and unknown metabolites. The primary and secondary metabolites observed in this study represent MVA, DOXP, shikimic acid and phenylpropanoid biosynthetic metabolic pathways. Squalene and tocopherol have been rated as the most potent naturally occurring compounds with antioxidant properties. These compounds have been identified by us for the first time in the fruits of W. somnifera. Multivariate principal component analysis (PCA) on GC-MS and NMR data revealed clear distinctions in the primary and secondary metabolites among the chemotypes. The variation in the metabolite concentration among different chemotypes of the fruits of W. somnifera suggest that specific chemovars can be used to obtain substantial amounts of bioactive ingredients for use as potential pharmacological and nutraceuticals agents.

Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet rhizospheres investigated by comparative transcriptomics.

BACKGROUND: The rhizosphere is the microbe-rich zone around plant roots and is a key determinant of the biosphere's productivity. Comparative transcriptomics was used to investigate general and plant-specific adaptations during rhizosphere colonization. Rhizobium leguminosarum biovar viciae was grown in the rhizospheres of pea (its legume nodulation host), alfalfa (a non-host legume) and sugar beet (non-legume). Gene expression data were compared to metabolic and transportome maps to understand adaptation to the rhizosphere. RESULTS: Carbon metabolism was dominated by organic acids, with a strong bias towards aromatic amino acids, C1 and C2 compounds. This was confirmed by induction of the glyoxylate cycle required for C2 metabolism and gluconeogenesis in all rhizospheres. Gluconeogenesis is repressed in R. leguminosarum by sugars, suggesting that although numerous sugar and putative complex carbohydrate transport systems are induced in the rhizosphere, they are less important carbon sources than organic acids. A common core of rhizosphere-induced genes was identified, of which 66% are of unknown function. Many genes were induced in the rhizosphere of the legumes, but not sugar beet, and several were plant specific. The plasmid pRL8 can be considered pea rhizosphere specific, enabling adaptation of R. leguminosarum to its host. Mutation of many of the up-regulated genes reduced competitiveness for pea rhizosphere colonization, while two genes specifically up-regulated in the pea rhizosphere reduced colonization of the pea but not alfalfa rhizosphere. CONCLUSIONS: Comparative transcriptome analysis has enabled differentiation between factors conserved across plants for rhizosphere colonization as well as identification of exquisite specific adaptation to host plants.

The biotransformation of aromatic amino acids by Phoma macrostoma.

A search for natural products produced in fermentation cultures of a strain of Phoma macrostoma led to the identification of tyrosol as the major metabolite produced by this organism. The addition of the amino acid tyrosine to growing fermentation cultures of P. macrostoma resulted in an increase in the production of tyrosol. It was subsequently determined that this strain of P. macrostoma was also capable of the biotransformation of other amino acids into similar alcohols.

Engineered native pathways for high kaempferol and caffeoylquinate production in potato.

Flavonols and caffeoylquinates represent important groups of phenolic antioxidants with health-promoting activities. The genetic potential of potato (Solanum tuberosum) to produce high levels of these dietary compounds has not been realized in currently available commodity varieties. In this article, it is demonstrated that tuber-specific expression of the native and slightly modified MYB transcription factor gene StMtf1(M) activates the phenylpropanoid biosynthetic pathway. Compared with untransformed controls, transgenic tubers contained fourfold increased levels of caffeoylquinates, including chlorogenic acid (CGA) (1.80 mg/g dry weight), whilst also accumulating various flavonols and anthocyanins. Subsequent impairment of anthocyanin biosynthesis through silencing of the flavonoid-3',5'-hydroxylase (F3'5'h) gene resulted in the accumulation of kaempferol-rut (KAR) to levels that were approximately 100-fold higher than in controls (0.12 mg/g dry weight). The biochemical changes were associated with increased expression of both the CGA biosynthetic hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (Hqt) gene and the upstream chorismate mutase (Cm) and prephenate dehydratase (Pdh) genes. Field trials indicated that transgenic lines produced similar tuber yields to the original potato variety Bintje. Processed products of these lines retained most of their phenylpropanoids and were indistinguishable from untransformed controls in texture and taste.

Aromatic amino acid metabolism during liver failure.

Liver failure is associated with hepatic encephalopathy (HE). An imbalance in plasma levels of aromatic amino acids (AAA) phenylalanine, tyrosine, and tryptophan and branched chain amino acids (BCAA) and their BCAA/AAA ratio has been suggested to play a causal role in HE by enhanced brain AAA uptake and subsequently disturbed neurotransmission. Until recently, data on this subject and the role of the liver and splanchnic bed were scarce, particularly in humans, due to inaccessibility of portal and hepatic veins. Here, we discuss, against a background of relevant literature, data obtained in patients undergoing liver resection or with a transjugular intrahepatic portasystemic stent shunt (TIPSS), where these veins are accessible. The BCAA/AAA ratio remained unchanged after major liver resection, but plasma AAA levels were inversely correlated (P < 0.001) with residual liver volume, in keeping with the observed hepatic AAA uptake. In patients with stable cirrhosis and a TIPSS, the plasma BCAA/AAA ratio was lower than in controls (1.19 +/- 0.09 vs. controls: 3.63 +/- 0.34). Gastrointestinal bleeding in cirrhotics with a TIPSS induced disturbances in BCAA levels and the BCAA/AAA ratio and induced catabolism, which could partly be corrected by isoleucine administration. AAA may be important in the pathogenesis of HE, but it is unlikely that they are the sole factors. HE most likely is a syndrome with multifactorial pathogenesis, where hyperammonemia, AAA/BCAA imbalances, inflammation, brain edema, and neurotransmitter changes interact. Novel therapies to normalize AAA levels in patients with liver failure (such as the molecular adsorbent recirculating system dialysis device) should probably be combined with supplementation of e.g. isoleucine and enhancing ammonia excretion by the kidneys.

Identification and functional characterization of a novel low affinity aromatic-preferring amino acid transporter (arpAT). One of the few proteins silenced during primate evolution.

We have identified in silico arpAT, a gene encoding a new member of the LSHAT family, and cloned it from kidney. Co-expression of arpAT with the heavy subunits rBAT or 4F2hc elicited a sodium-independent alanine transport activity in HeLa cells. L-tyrosine, l-3,4-dihydroxyphenylalanine (L-DOPA), L-glutamine, L-serine, L-cystine, and L-arginine were also transported. Kinetic and cis-inhibition studies showed a K(m) = 1.59 +/- 0.24 mM for L-alanine or IC50 in the millimolar range for most amino acids, except L-proline, glycine, anionic and D-amino acids, which were not inhibitory. L-DOPA and L-tyrosine were the most effective competitive inhibitors of L-alanine transport, with IC50 values of 272.2 +/- 57.1 and 716.3 +/- 112.4 microM, respectively. In the small intestine, arpAT mRNA was located at the enterocytes, in a decreasing gradient from the crypts to the tip of the villi. It was also expressed in neurons from different brain areas. Finally, we show that while the arpAT gene is conserved in rat, dog, and chicken, it has become silenced in humans and chimpanzee. Actually, it has been recently reported that it is one of the 33 recently inactivated genes in the human lineage. The evolutionary implications of the silencing process and the roles of arpAT in transport of L-DOPA in the brain and in aromatic amino acid absorption are discussed.

Aromatic amino acid transporter AAT-9 of Caenorhabditis elegans localizes to neurons and muscle cells.

The Caenorhabditis elegans genome encodes nine homologues of mammalian glycoprotein-associated amino acid transporters. Two of these C. elegans proteins (AAT-1 and AAT-3) have been shown to function as catalytic subunits (light chains) of heteromeric amino acid transporters. These proteins need to associate with a glycoprotein heavy chain subunit (ATG-2) to reach the cell surface in a manner similar to that of their mammalian homologues. AAT-1 and AAT-3 contain a cysteine residue in the second putative extracellular loop through which a disulfide bridge can form with a heavy chain. In contrast, six C. elegans members of this family (AAT-4 to AAT-9) lack such a cysteine residue. We show here that one of these transporter proteins, AAT-9, reaches the cell surface in Xenopus oocytes without an exogenous heavy chain and that it functions as an exchanger of aromatic amino acids. Two-electrode voltage clamp experiments demonstrate that AAT-9 displays a substrate-activated conductance. Immunofluorescence shows that it is expressed close to the pharyngeal bulbs within C. elegans neurons. The selective expression of an aat-9 promoter-green fluorescent protein construct in several neurons of this region and in wall muscle cells around the mouth supports and extends these localization data. Taken together, the results show that AAT-9 is expressed in excitable cells of the nematode head and pharynx in which it may provide a pathway for aromatic amino acid transport.

Overexpression of a cyanobacterial phosphoenolpyruvate carboxylase with diminished sensitivity to feedback inhibition in Arabidopsis changes amino acid metabolism.

Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Synechococcus vulcanus (SvPEPC) is a unique enzyme, being almost insensitive to feedback inhibition at neutral pH. In order to assess its usefulness in metabolic engineering of plants, SvPEPC was expressed in Arabidopsis thaliana (L.) Heynh. under the control of the cauliflower mosaic virus 35S promoter. About one-third of the transformants of the T1 generation showed severe visible phenotypes such as leaf bleaching and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with Zea mays L. PEPC (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, L-malate. For the SvPEPC transformants of the T2 generation, which had been derived from fertile T1 transformants, three kinds of phenotype were observed when plants were grown on an agar medium containing sucrose: Type-I plants showed poor growth and a block in true leaf development; Type-II plants produced a few true leaves, which were partially bleached; Type-III plants were apparently normal. In Type-I plants, total PEPC activity was increased about 2-fold over the control plant but there was no such increase in Type-III plants. The phenotypes of Type-I plants were rescued when the sucrose-containing agar medium was supplemented with aromatic amino acids. Measurement of the free amino acid content in whole seedlings of Type-I transformants revealed that the levels of the aromatic amino acids Phe and Tyr were lowered significantly as compared with the control plants. In contrast, the levels of several amino acids of the aspartic and glutamic families, such as Asn, Gln and Arg, were markedly enhanced (4- to 8-fold per plant fresh weight). However, when the medium was supplemented with aromatic amino acids, the levels of Asn, Gln, and Arg decreased to levels slightly higher than those of control plants, accompanied by growth recovery. Taken together, it can be envisaged that SvPEPC is capable of efficiently exerting its activity in the plant cell environment so as to cause imbalance between aromatic and non-aromatic amino acid syntheses. The growth inhibition of Type-I plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate, one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. The possible usefulness of SvPEPC as one of the key components for installing the C4-like pathway is proposed.

Brain amino acid abnormalities in liver disease--a postmortem study.

In a postmortem exploratory study, we examined whether specific amino acid abnormalities associated with liver diseases in vivo may also be detected in human brain samples obtained at clinical autopsies. The branched-chain amino acids (BCAA: valine, leucine, isoleucine) were decreased in the group of patients with liver diseases compared with the control group, whereas the aromatic amino acids (AAA: phenylalanine, tyrosine) were increased. However, the ranges overlapped significantly and were not statistically different. The molar ratio BCAA/AAA was determined to be 1.92 in the collection of patients with liver diseases compared with 2.27 in the control group. In patients with liver disease, ornithine concentrations in the brain appeared significantly decreased whereas glutamine was significantly increased. No significant difference was found in the brain concentrations of proline. Amino acid analysis may contribute to the understanding of pathophysiological mechanisms of liver disease, which are discussed, and may supplement the postmortem diagnosis.